RESUMEN
Yeast two-hybrid (Y2H) systems are one of the principal choices for identifying novel binary protein-protein interactions (PPIs). Since its development, it has contributed for the discovery of several PPIs between pathogens and host, allowing not only a comprehensive look at the disease pathogenesis but also for therapeutic strategies. Identification of viral-host PPIs that impact on viral replication and pathogenesis can lead to new advances in antiviral therapies such as the development of drug candidates and vaccine design. In this chapter, we revise the Y2H key parameters necessary for screening PPIs and discuss the possible approaches for using this technique to identify novel dengue-host protein interactions.
Asunto(s)
Virus del Dengue , Dengue , Humanos , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , Replicación ViralRESUMEN
A growing body of evidence demonstrates that endothelial cells (ECs) play a prominent role in immune-enhanced pathology seen in dengue virus (DENV) infection that might contribute to vascular permeability and hemorrhagic manifestations in severe dengue cases. However, it remains a question of whether DENV infection of ECs directly causes permeability or if extra-endothelial factors such as immune cell activation or antibody-dependent enhancement (ADE) are required. In this chapter, we detail the measurement of the transendothelial electrical resistance (TEER), a quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial monolayers and show that DENV infection of ECs does not cause endothelial permeability in vitro.
Asunto(s)
Virus del Dengue , Dengue , Permeabilidad Capilar , Impedancia Eléctrica , Células Endoteliales , HumanosRESUMEN
RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by â¼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.
Asunto(s)
Fosfatos/metabolismo , ARN/metabolismo , Aptámeros de Nucleótidos , Línea Celular , Humanos , Cinética , Límite de Detección , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Proteínas/metabolismo , Estabilidad del ARN , Estándares de Referencia , Suero/metabolismo , Termodinámica , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE: Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Virus del Dengue/metabolismo , Dengue/metabolismo , Dengue/virología , Vitronectina/metabolismo , Línea Celular Tumoral , Flavivirus/metabolismo , Humanos , Unión Proteica/fisiología , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/metabolismo , Virus Zika/metabolismo , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/virologíaRESUMEN
Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.